1. Field of the Invention
In plants, acyl-carrier protein (ACP) exists as a small acidic cofactor protein which participates in at least 12 reactions of fatty acid biosynthesis and metabolism. In recent years, research on this protein has intensified in several laboratories because of the potential of ACP to serve as a representative marker protein for studies of the regulation of plant fatty acid synthetase gene expression. Such studies may eventually have an important practical impact on the selection of genetic engineering strategies used to modify the amount and type of fatty acids produced by oilseed crops. For example, ACP levels have been measured in developing soybean seeds by both enzymic and immunochemical assays [J. B. Ohlrogge et at. I, Plant Physiol. 74: 622-625 (1984)]. A close correlation was found between rates of fatty acid synthesis in vivo and ACP content. These results suggest that levels of fatty acid biosynthetic proteins may be a rate-determining component of the seed's overall lipid biosynthetic capacity. Although other factors such as substrate and cofactor supply may also limit seed oil production, the results with ACP provide encouragement that molecular genetic modification of fatty acid biosynthetic protein levels may provide a means to influence oilseed metabolism.
2. Description of the Prior Art
ACPs have been the first proteins in plant fatty acid biosynthesis to be purified to homogeneity and, to date, the only proteins for which amino acid sequence data are available. Spinach leaf ACP-I has been completely sequenced [T. M. Kuo et al. I, Arch. Biochem. Biophys. 234: 290-296 (1984)], and 72 of 87 residues of the barley leaf ACP-I are known [P. B. Hoj et al., Carlsberg Res. Commun. 48: 284-306 (1983)]. The two plant sequences are 70% homologous, indicating that the ACP structure is highly conserved between monocot and dicot plant species. Comparison with the Escherichia coli ACP sequence reveals 40% homology; whereas, the ACP domain of the rabbit multi-enzyme fatty acid synthetase complex has 25% homology with plant or bacterial ACP sequences. These comparisons suggest that all ACPs evolved from a common ancestor, but, intriguingly, the plant structure has remained closer to its bacterial counterpart than to the corresponding animal structure.
Plants have recently been shown to contain multiple isoforms of ACP [P. B. Hoj et al., Carlsberg Res. Commun. 49: 483-492 (1984); J. B. Ohlrogge et al. II, J. Biol. Chem. 260: 8032-8037 (1985)]. Although the isoforms are clearly closely related in structure, there are significant differences in the amino acid composition and the N-terminal sequences of both barley and spinach ACP isoforms. These differences suggest that the isoforms may be coded by multigene families.
The plant ACP isoforms are expressed differently in different tissues (Ohlrogge et al. II, supra). In spinach leaves we find that ACP-I is present at three- to fourfold higher levels than ACP-II. However, in developing spinach seeds ACP-II is the predominant species, with ACP-I absent or barely detectable. Similar results have been observed with castor oil seed leaves and endosperm and soybean leaves and developing cotyledons.
Studies have revealed that ACP is localized essentially exclusively in the plastids of spinach mesophyll cells, but is probably initially synthesized in the cytoplasm. Reported data also suggest that ACP is a nuclear-encoded protein, which is synthesized as a precursor polypeptide containing a transit peptide that guides its uptake by the plastids.
ACPs constitute less than 0.1% of the total cell protein in most species [T. M. Kuo et al. II, Arch. Biochem. Biophys. 230: 110-116 (1984)]. Therefore, purification of milligram quantities is difficult, and, as a consequence, plant lipid biosynthetic studies have been hampered by the absence of adequate supplies of plant ACP for use as cofactor or substrate.
Expression of a plant ACP gene in a suitable vector such as E. coli might provide a means of providing sufficient ACP for enzymological and other studies. A synthetic gene encoding only a strutural protein is more likely to produce an active ACP when introduced into E. coli than either a genomic clone (with expected intervening sequences, i.e., introns) or a full-length cDNA clone (with an expected transit peptide encoding sequence).